Characterization of Ras Y4H mutants in Drosophila

Ras signaling plays a highly conserved role from flies to mammals in establishing proper development, and its dysregulation can lead to cancer. In Drosophila , we demonstrated that Ras Tyrosine 4 (Y4) was required for inhibitory ubiquitination by Rabex-5. In humans, rare histidine substitution mutations at Y4 are found in HRas in cerebellar glioblastomas (cGBMs). We report here that analogous Y4H mutations in Drosophila Ras make it less sensitive to Rabex-5-mediated ubiquitination in cells and show increased frequency of vein phenotypes per wing compared to wild-type Ras, which would be consistent with Ras gain-of-function and with their appearance in human cGBMs.

(A) S2 cells were transfected with FLAG-HIS6 tagged Ras WT or Ras Y4H and HA-Ubiquitin.Western blots were stained with anti-HA (to label ubiquitin conjugates, upper panels) or anti-FLAG (to label Ras and Ras ubiquitin conjugates, lower panels).Ras WT showed increased ubiquitination when co-transfected with Rabex-5 but Ras Y4H did not.Mono-and di-ubiquitin conjugates in both sets of gels are labeled to the right with an asterisk.Quantification of anti-FLAG gels shows percent of Ras in ubiquitin conjugates in graph under the Western panels.**** indicates p<0.0001 and ns indicates not significant in Fisher's exact test and Chi-square tests as calculated in GraphPad Prism.(B) Schematic of a wing labeling L2, L3, L4, and L5 longitudinal veins in a wing with normal morphology.(B') Control ms1096-gal4 wing.(C-E) ms1096-gal4 driving expression of one copy of (C) UAS Ras WT , (D) UAS Ras Y4F , and (E) UAS Ras Y4H .Under conditions where Ras WT expression causes very little phenotype, Ras Y4F and Ras Y4H expression both cause increased wing vein abnormalities associated with Ras gain-offunction.Abnormalities are most notable between L2 and the wing margin, between L2 and L3, and where L4 and L5 meet with wing margin (examples of these specific phenotypes are indicated by arrowheads in 1D).(F) We scored all wings expressing one copy of the indicated transgene (or ms1096-gal4 control) for the presence of phenotypes between L2 and the wing margin (L2-WM), between L2 and L3 (L2-L3), where L4 meets the wing margin (L4), or where L5 meets the wing margin (L5).The left graph indicates the percentage of wings in a typical trial with each of these phenotypes per genotype.In this trial, there is a clear difference between genotypes for L2-L3, but this was not true in every trial; we saw variability between trials for specific phenotypes.We also quantified the number of phenotypes per wing (no phenotypes, 1 or more, 2 or more, or 3 or more phenotypes.The right graph indicates the percentage of wings in each of these categories per genotype.Despite the variability at specific vein positions, reproducibly all three trials scored were highly reproducible for the increased frequency of phenotypes.We saw statistically significant differences between Ras WT and Ras Y4H using Chi-square and Fisher's exact tests in Graphpad Prism when comparing categories of no phenotypes versus 1 or more phenotypes or 0-1 phenotype versus 2 or more phenotypes (see Statistical analysis section in Methods for p values and details).Although we reproducibly saw differences between categories of less than 3 phenotypes versus 3 or more phenotypes per wing, this was not always statistically significant possibly due to the lower number of wings with 3 or more phenotypes.(G-I) ms1096-gal4 wings homozygous for (G) UAS Ras WT , (H) UAS Ras Y4F , and (I) UAS Ras Y4H .Male wings are shown in B-H.Female wings of the same genotypes show weaker phenotypes but the same phenomena.

Description
Ras (called Ras85D and Ras1 in Drosophila, here referred to as "Ras") is an important regulator of development and is dysregulated in Rasopathies and cancer.Ras is represented by one gene in Drosophila and by HRas, NRas, and KRas in mammals.Drosophila Ras and HRas and NRas are negatively regulated by inhibitory ubiquitination [Jura et al., 2006;Yan et al., 2009] by the E3 Rabex-5 [Yan et al. 2010, Xu et al 2010].We previously showed that Rabex-5 requires Tyrosine 4 (Y4) to promote Ras ubiquitination in Drosophila [Washington et al., 2020]; Y4F mutations cause Ras to evade ubiquitination by Rabex-5 in vitro and behave as gain-of-function Ras mutations in vivo [Washington et al. 2020].
Curiously, Y4 mutations in HRas, HRas Y4H , have been reported in cerebellar glioblastoma multiforme (cGBM) [Milinkovic et al., 2014], a rare form of glioblastoma multiforme (GBM).Because our previous work demonstrated the importance of Y4 for Ras recognition by Rabex-5 [Washington et al., 2020], we speculated that these Y4H mutations might act like gain-of-function mutations by evading inhibition by Rabex-5 to aid in cerebellar glioblastoma growth.We engineered Y4H mutations in Drosophila Ras, Ras Y4H , in pIE 1-4 vectors for tissue culture work and pUASTattb for generating transgenic lines with sitespecific integration.Ras Y4H was tagged N-terminally with FLAG-HIS6 tags used for wild type Ras, Ras WT , and Ras Y4F as in our previous work [Washington et al., 2020].Transgenic lines used insertion at the attp40 site, the same genomic site as previously generated lines UAS Ras WT and UAS Ras Y4F , so any difference in phenotype would be due to the specific Y4 mutation and not position insertion effects.
We isolated Ras ubiquitin conjugates from cells using previous protocols [Yan et al., 2009;Yan et al., 2010;Washington et al., 2020].S2 cells were transfected with Ras constructs and HA-UB plasmid with vector control or Rabex-5, and Ras was pulled down on nickel beads and then analyzed by Western.Co-transfection of S2 cells with Rabex-5 increased the ubiquitination of Ras WT above baseline to a far greater extent than seen with Ras Y4H (Fig. 1A).In vivo, expressing Ras WT using ms1096-gal4 results in wings with some extra wing vein material (Fig. 1C) compared to the normal vein pattern of control wings (Fig. 1B', schematic of wing veins shown in Fig. 1B) most notably disrupted or ectopic vein material where L4 meets the wing margin but also some frequency of disrupted or ectopic vein material between L2 and the wing margin, between L2 and L3, and disrupted or ectopic vein material where L5 meets the wing margin.As we reported previously [Washington et al., 2020], expressing Ras Y4F causes increased abnormalities compared to Ras WT (Fig. 1D, quantified in Figure 1F).Expressing Ras Y4H resembles Ras Y4F and shows increased abnormalities compared to Ras WT (Fig. 1E, quantified in Figure 1F).Because these wing phenotypes are variable, to establish the increase in wing vein abnormalities, three different lab members scored male wings expressing one copy of each transgene with ms1096-gal4 compared to control ms1096-gal4 wings for the presence or absence of a phenotype at four sites: between L2 and the wing margin, between L2 and L3, where L4 meets the wing margin, and where L5 meets the wing margin (Fig. 1F left graph, phenotypes evident in wings shown in Fig. 1D-1E, 1G-1I and noted by arrowheads in 1D).We see some background of phenotypes in control wings, and the incidence of phenotypes increases upon Ras WT expression as is seen throughout the literature.We saw variability in the incidence of specific phenotypes between trials, so we cannot conclude that Ras Y4F and Ras Y4H cause greater frequency of the L2-L3 phenotype as shown in the trial depicted in Fig. 1F.We did not see reproducible differences in severity between Ras Y4F and Ras Y4H .However, reproducibly in the three data sets scored, the number of phenotypes per wing statistically significantly increases across the population for Ras Y4H compared to Ras WT (Fig. 1F, right graph).Taken together, the failure of Rabex-5 to increase Ras Y4H ubiquitination in vitro to the extent it did for Ras WT (Fig. 1A) and the increased frequency of phenotypes per wing of Ras Y4H in vivo compared to Ras WT (Fig. 1F) are consistent with a model that Ras Y4H acts as a gain-of-function Ras mutation by evading Rabex-5 inhibition.Increasing the gene dosage by generating flies homozygous for the Ras transgenes showed stronger wing vein phenotypes; subjective observations by independent observers scored these phenotypes as stronger across the population for Ras Y4F (Fig. 1H) and Ras Y4H (Fig. 1I) compared to Ras WT (Fig. 1G), however relative severity of this was not quantified due to the more subjective nature of such scoring.Future work will define corresponding increases in molecular readouts of Ras activity, such as an increase in the relative proportion of dpERK, and will explore the conservation of this result to elucidate if HRas Y4H variants identified in cGBMs underlie the etiology of cGBMs by also evading Ras ubiquitination to act as Ras gain-of-function mutations.

Schneider S2 cell extract preparation and Nickel pull down of tagged Ras from Schneider S2 cells (for Western analysis):
Schneider S2 cell extracts were prepared and used to isolate tagged Ras in Figure 1A as described previously [Washington et al., Yan et al. 2009;Yan et al 2010].Extracts were prepared from harvested cells or from frozen cell pellets in lysis buffer, 1X NP40 buffer (50 mM HEPES pH 7.4, 1% NP40, 1mM EDTA, 150 mM NaCl), 8M Urea, 5 mM betamercaptoethanol, 10 mM imidazole supplemented with Roche protease inhibitor cocktail and 1mM each of PMSF and Sodium Orthovanadate) and used in pull down assays described above or analyzed by Western.Ni-NTA agarose beads (Qiagen) were equilibrated per manufacturer's instructions, washed twice with binding buffer, pH 8.0 (1X PBS, 8 M Urea, 0.5% NP40, 10 mM Imidazole, 360 mM NaCl), then incubated with binding buffer, pH 8.0 supplemented with 1g/ml BSA.Lysates were incubated with beads at 4°C for 2 hours.Beads were transferred to mini-columns on a vacuum assembly and washed 3X with wash buffer pH 6.3 (1X PBS, 8 M Urea, 0.5% NP40, 30 mM Imidazole, 360 mM NaCl).Proteins were eluted from the beads with elution buffer pH 6.3 (1X PBS, 8 M Urea, 0.5% NP40, 500 mM Imidazole, 360 mM NaCl), boiled in 1X loading buffer for 10 minutes, then stored at -20°C or run immediately on a gel.

Quantification of ubiquitination.
Quantification of percent ubiquitination utilized gels in which signals were not oversaturated.To quantify percent ubiquitination, gel lanes were analyzed using Image J; we summed the signal of unconjugated and ubiquitinated Ras bands in each lane to define total Ras signal in that lane and then calculated the proportion of each band per total to indicate percent of Ras conjugated to ubiquitin out of total Ras using a method used previously [Washington et al., 2/29/2024-Open Access 2020].In this manner, we could make comparisons of percent conjugation to ubiquitin in one lane to the percent conjugation to ubiquitin in another lane from different lanes on the same gel within the same experiment.

Statistical analysis comparing ubiquitin conjugates.
Statistical analysis comparing the percent of conjugated and unconjugated Ras shown in Figure 1A (as described above) used Fisher's exact test and Chi-square tests in Graphpad Prism.
Statistical analysis comparing the incidence of wing vein phenotypes.We compared categories of no phenotypes versus 1 or more phenotypes; 0 and 1 phenotype versus 2 or more phenotypes; or less than 3 phenotypes versus 3 or more phenotypes per wing in control ms1096-gal4 wings compared to ms1096-gal4 driving Ras transgenes or of ms1096-gal4 driving Ras WT compared to ms1096-gal4 driving Ras Y4H with both Chi-square and Fisher's exact tests in Graphpad Prism.We saw statistically significant differences between all three transgenes and control ms1096-gal4 wings when comparing no phenotypes versus 1 or more (p<0.0001for all cases), when comparing 0-1 phenotype versus 2 or more phenotypes (p=0.0008 in Chi-square and p=0.0005 in Fisher's exact test for control ms1096-gal4 versus Ras WT and p<0.0001 for control ms1096-gal4 versus each Y4 mutant).We saw no difference between control ms1096-gal4 and Ras WT when comparing less than 3 phenotypes versus 3 or more phenotypes (p=0.0609 in Chi-square and p=0.0873 in Fisher's exact test), but statistically significant difference when comparing control ms1096-gal4 to Ras Y4F (p=0.0013 in Chi-square and p=0.0035 in Fisher's exact test) or Ras Y4H (p=0.0021 in Chi-square and p=0.0020 in Fisher's exact test).We saw statistically significant differences between Ras WT and Ras Y4H using Chi-square and Fisher's exact tests in Graphpad Prism when comparing categories of no phenotypes versus 1 or more phenotypes (p=0.0175 in Chi-square test and p=0.0260 in Fisher's exact test) or 0-1 phenotype versus 2 or more phenotypes (p=0.029 in Chi-square test and p=0.0175 in Fisher's exact test).Although we reproducibly saw differences between categories of less than 3 phenotypes versus 3 or more phenotypes per wing, this was not always statistically significant possibly due to the lower number of wings with 3 or more phenotypes.
Drosophila experiments.UAS Ras Y4H was cloned into pUAST-attB with the FLAG and His6 sequences of MDYKDDDDKRGSHHHHHHALE preceding the Ras coding sequence as we did previously with UAS FLAG-His6-Ras WT  and UAS Flag-His6-Ras Y4F .The plasmid was sent to BestGene for injection and generation of transgenic lines at the attp40 locus.Lines were balanced over CyO or SM6-TM6B balancers and then maintained as true-breeding homozygous stocks.Genomic DNA was sequenced to confirm each insert.Importantly, UAS Ras Y4H differs in sequence from UAS Ras Y4F and UAS Ras WT only at Y4. Crosses were performed at the indicated temperatures on standard Drosophila medium.Raw wing images were converted to grayscale using Adobe Photoshop.Brightness and contrast of wing images were adjusted using Adobe Photoshop to maximize clarity; adjustments were applied to the entire images.Genotypes are summarized below, and identifiers are annotated in Table 1.Protein sequences for Ras transgenes are detailed in Table 2.

Figure 1 .
Figure 1.Ras Y4H is insensitive to Rabex-5-mediated ubiquitination and shows increased frequency of wing vein phenotypes compared to Ras WT in vivo.: